Autoimmune disease (AD) affects up to 50 million Americans, which costs approximately $100 billion on health care annually and accounts for one of the leading causes of death in women. T cells and cytokine function play an important role in autoimmune diseases, such as Type-1 Diabetes, Multiple Sclerosis, Rheumatoid Arthritis (RA), and Systemic Lupus Erythematosus (SLE). The most successful auto-immune therapies already target the factors secreting from or binding to T cells, e.g., TNF-?, IL-6, IL-1, and IL-15. Diagnosis of autoimmune diseases is typically made post presentation of symptoms; enabling earlier diagnosis through detecting the T cells that drive disease progression (pathogenic T cells) could shift paradigms towards earlier and more successful treatment. IsoPlexis is developing a system to target and measure single T cells integral to this early diagnosis. In this Phase I project, we will use SLE as a preliminary disease with which to evaluate the strength of our technology. SLE is an autoimmune disease mediated by autoreactive antibodies, yet a subset of T cells called follicular helper T (Tfh) cells, when dysregulated or pathogenic, promote the induction of B cell autoantibody production. Identifying these rare, disease-causing T cells, also present in circulation, has been proposed as a critical early diagnostic and a pressing need in the clinic. However, there are major challenges that need to be addressed: (1) pathogenic, circulating Tfh cells are present at low frequency in blood, (2) these cells are highly heterogeneous with diverse combinations of effector/cytokine functions, requiring single-cell analysis, and (3) the range of cytokine functional groups that matters numbers ~15+. The IsoPlexis technology addresses these unmet challenges by (1) simultaneously assaying ~1500 cells, (2) analyzing cell functionality at the single-cell level, and (3) co-detecting ~30 effector proteins per cell. This SBIR application aims to develop a suitable solution, and an alpha test prototype device, by incorporating an on-chip, upstream, cell sorting method for capturing low quantities of circulating Tfh cells, followed by a validated, single-cell, cytokine function profiling platform for identifying pathogenic T cells reproducibly. This NIH Phase I SBIR project will focus on developing this assay and device for lupus and autoimmune research. We propose the following specific aims: (1) develop an NACS-based cell capture device for profiling circulating Tfh cells with SCBC technology to eliminate flow sorting, and establish a single-cell, 30-plex, cytokine and chemokine panel specific to profiling Tfh cells; and (2) develop the first components of an integrated automation workflow system for capturing and analyzing single Tfh cells with required reproducibility. With a validated test system, we would, in Phase II, apply this test to a large cohort clinical study and fully validate this for further commercialization in a critical area for SLE patients and physicians. If successful, this will open new opportunities for autoimmune disease diagnosis and monitoring of other T cell cytokine mediated diseases.